1. Field of Invention
This invention relates to organ preservation and hypothermic blood substitution. This invention particularly relates to compositions, processes and systems for organ and tissue preservation and/or hypothermic blood substitution.
2. Description of Related Art
Hypothermia is the bed rock of all useful methods of organ and tissue preservation, and has proven to be most effectively applied by controlling the extracellular environment of cells directly, and the intracellular environment indirectly, during cold exposure. Control of the extracellular environment of cells to optimise preservation is based upon different strategies that include either static cold storage (or flush preservation), or low temperature continuous perfusion. These different strategies call for different approaches to interventional control of the extracellular environment in order to optimize preservation, and hence different design elements for the solutions used to effect these strategies.
In principle, cold flush storage or preservation is based upon the premise that temperature reduction to near but not below the ice point (0° C.) precludes the need to support metabolism to any significant extent, and that the correct distribution of water and ions between the intracellular and extracellular compartments can be maintained by physical rather than metabolic means. During the period that metabolic pumps are inactivated, the driving force for transmembrane ion flux is the difference in ionic balance between intracellular and extracellular fluid. The driving force for water uptake (cell swelling) is the impermeant intracellular anions. Thus changes can be prevented or restricted by manipulating the extracellular environment to abolish chemical potential gradients. On this basis, a variety of flush, or organ washout, solutions have been devised and evaluated for cold storage. These solutions are often referred to as “intracellular” solutions due to their resemblance, in some respects, to intracellular fluid.
The principle design elements of the “intracellular” flush solutions has been to adjust the ionic balance (notably of the monovalent cations) and to raise the osmolality by including an impermeant solute to balance the intracellular osmotic pressure responsible for water uptake. However, the most important factor for the efficacy of cold flush solutions may be the prevention of cellular edema by inclusion of impermeant solutes since it has been established that ionic imbalances, especially potassium depletion, are readily and rapidly reversible.
Prior to 1988, the standard solution for clinical preservation of abdominal organs, principally the kidney, was Collins solution, which consists predominantly of potassium phosphate, magnesium sulfate and glucose. In recent years, however, this has been superseded either by a modified version called “EuroCollins” in which the magnesium sulfate is omitted, or more extensively by the University of Wisconsin solution (UW solution) in which much of the phosphate anion has been replaced with lactobionate, and in which glucose has been replaced with raffinose. These larger molecules provide better protection against adverse effects of cell swelling during hypothermic storage. The choice of solutions for heart preservation has been strongly influenced by the previous experience of cardiac surgeons with cardioplegic solutions in open-heart surgery. In this case the primary objective has been to produce rapid cessation of the heartbeat, and solutions were designed more with this in mind than with protection of the cells during preservation in mind. In particular, early studies suggested that the very high potassium levels (>100 mM) found in organ preservation solutions might be harmful to the heart. In fact, the solution most often used was St. Thomas's (Plegisol) with a potassium content of only 16 mM.
The choice of solution for cardioplegia and myocardial preservation remains controversial and widely varied. While UW solution has emerged as the industry standard for kidney, liver and pancreas, no such standard has been adopted for heart preservation. Moreover, the development of the variety of preservation solutions for organ storage has emphasized the need for careful optimization in relation to the specific characteristics of the tissue to be preserved.
Attention to biophysical properties of “intracellular” flush solutions, to restrict passive diffusional processes, has unquestionably led to the development of techniques that have provided the basis of clinical organ preservation during the past 30 years. Nevertheless, it is recognized that further optimization of cold flush solutions can be achieved by inclusion of biochemical and pharmacological components that will be effective in counteracting the deleterious effects of ischemia and reperfusion injury. To a limited extent this approach has been incorporated in the design of the University of Wisconsin organ preservation solution (UW solution marketed as “Viaspan™”; DuPont) which has become the most widely used solution for cold flush preservation of kidneys, livers and pancreases. With due consideration for the effects of ischemia, hypoxia, hypothermia and reperfusion injury on cells, coupled with the proven efficacy of various existing organ preservation solutions, a general consensus of the most important characteristics in the design of hypothermic storage solutions has emerged. These include: minimizing of hypothermically induced cell swelling; preventing expansion of the interstitial space (especially important during perfusion); restricting ionic imbalances; preventing intracellular acidosis; preventing injury from free radicals; and providing substrates for regeneration of high energy phosphate compounds during reperfusion.
In continuous hypothermic perfusion preservation, the desirable properties of hypothermic solutions listed above are also applicable to controlling the extracellular environment by way of continuous perfusion techniques. In contrast to static cold storage, continuous perfusion is usually controlled at around 10° C. and is based upon a different principle: it is generally assumed that a moderate degree of cooling will reduce metabolic needs but that continuous perfusion is required to support the suppressed metabolism and remove catabolic products. Because it is assumed that sufficient metabolic activity remains to actively regulate a near-normal cell volume and ionic gradients, the perfusates are generally acellular, isotonic, well oxygenated solutions having a composition that more closely resembles plasma than intracellular fluid. Such perfusates are therefore designated as “extracellular” solutions, and are perfused through the vascular bed of an organ at a pressure sufficient to achieve uniform tissue distribution (typically 40–60 mm Hg). To balance this applied hydrostatic pressure and prevent interstitial edema, oncotic agents such as albumin or synthetic macromolecular colloids are incorporated into the perfusates. Substrate support of the remaining metabolism at ˜10° C. is also an important consideration and it has been shown in several organs that high energy adenine nucleotides can be synthesized during hypothermic perfusion preservation.
In addition to the principal objective of supporting metabolism, continuous perfusion also provides other advantages over flush preservation. These include the wash out of accumulated lactate and protons, thereby removing the metabolic block on glycolysis; this is thought to be especially beneficial for organs that have suffered prior warm ischemia. Perfusion also facilitates the removal of erythrocytes from the microcirculation and helps to maintain vascular patency during prolonged storage. Continuous perfusion has been shown to provide the best means of achieving prolonged hypothermic preservation (e.g., 3–7 days for kidneys), but concerns for damage to the vascular endothelium during prolonged perfusion may be a limiting factor.
Although it has been experimentally verified that cell metabolism continues at temperatures as low as 10° C., and that adenine nucleotides can be resynthesized during hypothermic preservation if appropriate substrates are provided, it is considered unlikely that this level of metabolism can prevent transmembrane ion and water movements: this is due principally to the temperature sensitivity of the active pumps. Hence, some advocates of continuous perfusion have modified the perfusate accordingly by increasing both the K+ concentration and the osmolality. Similarly, modification of cold flush solutions can be considered to circumvent some of the identified limitations of that approach. For example, the lack of support of metabolism during ice-storage can be addressed by raising the temperature of storage, by incorporating biochemical substrates and raising the oxygen tension to promote adenine nucleotide repletion. Also, the use of pharmacological agents, such as inhibitors of 5′-nucleotidase (e.g., allopurinol) has been advocated as a means of averting adenine nucleotide depletion.
With respect to specific cell requirements as a function of temperature, it is not necessary to consider incorporating specific oxygen-carrying molecules at temperatures below ˜10° C. since at such low temperatures it is well established that metabolic activity is sufficiently depressed that the O2 demand can be satisfied by dissolved O2 in the aqueous solution without the need for hemoglobin or synthetic O2-carrying molecules.
Optimum control of the intracellular and extracellular environment of cells during hypothermia depends upon the interaction of a variety of factors that include temperature, oxygen tension, acidity, osmotic pressure and chemical composition of the perfusion fluid or wash-out solution.
It is now recognized that the successive phases of the transplantation procedure involving organ procurement, storage, transportation, reimplantation and reperfusion may impose different requirements for optimum preservation at the different stages. This is illustrated by evidence that heart preservation with the “intracellular” solution, EuroCollins, was enhanced when the heart was initially arrested and subsequently flushed prior to reperfusion, with an “extracellular” cardioplegic solution. Therefore, any single formulation of preservation solution is unlikely to provide optimum protection during all the processing stages of a transplantation procedure, or the interventional stages of complex surgeries.
Interest in general or universal tissue preservation techniques is exemplified by the need for methods of protecting multiple vital organs, and even the whole body, for applications in modem day surgery. Multiple organ harvesting for transplantation can be optimized by hypothermic perfusion of the whole cadaver, or donor organ blocks comprising several organs, to minimize warm ischemic injury. The ultimate challenge is perhaps protection of the entire body against the effects of global ischemia during periods of circulatory and/or cardiac arrest for “bloodless” surgery.
Surgeons have developed skills that allow very complex, corrective and life-saving operations to be performed, notably on the heart and brain. Many of these complicated time-consuming procedures have the inherent need for temporary cessation of blood flow and demand protection of the patient against the deleterious effects of ischemia and anoxia. Although hypothermia is routinely used as an adjunctive protective modality for surgical procedures that require a period of cardiac arrest, there are restrictive time constraints (<1 hour at temperatures usually not lower than 18° C.) upon the safe interval of cold ischemia if neurological sequelae are to be avoided. It is well recognized that the window of opportunity for safe surgical intervention could be extended by using greater degrees of hypothermic metabolic suppression, but this becomes unacceptably dangerous due principally to the effects of profound hypothermia on the blood, leading to coagulopathies and irreversible microvascular blockage.
U.S. Pat. Nos. 5,643,712, 5,699,793, 5,843,024 to Brasile and U.S. Pat. Nos. 5,599,659, 5,702,881 to Brasile et al., each of which is incorporated herein by reference in its entirety, describe separate resuscitation and preservation solutions for tissues and organs. The Brasile patents disclose methods in which some embodiments of the compositions of this invention can be applied. Also, the Brasile patents disclose compositions that may be used in methods and kits of this invention.
The present inventor has explored experimental approaches employing a technique of asanguineous blood substitution using acellular synthetic solutions designed to protect the heart, brain and visceral organs during several hours of bloodless perfusion. The concept of using ultraprofound hypothermia (<10° C.) and complete blood replacement is appealing for several reasons and is based upon a variety of factors. First, deeper hypothermia can provide more effective suppression of metabolism, thereby extending the tolerance to ischemia and minimizing the demand for oxygen to levels that can be adequately supplied in a cold aqueous solution without the need of special oxygen-carrying molecules. Second, complete exsanguination ameliorates a complication associated with increased viscosity, coagulopathies, and erythrocyte clumping of cooled blood. Third, vascular purging can remove harmful catabolic products and formed elements that might participate in the ischemia and reperfusion injury cascades. A fourth advantage is that total exsanguination provides the opportunity to control the vascular and extracellular compartments directly with fluids designed to be protective under the conditions of ultraprofound hypothermia. For example, solutes can be added to maintain ionic and osmotic balance at the cellular and tissue levels; biochemical and pharmacological additives can help sustain tissue integrity in a variety of ways including efficient vascular flushing, membrane stabilization, free-radical scavenging and providing substrates for the regeneration of high-energy compounds during rewarming and reperfusion. In essence, these are the principles that are embodied, to a greater or lesser extent, in the design of various solutions used for ex vivo organ preservation. In this invention, similar principles have been adopted in the design of new hypothermic blood substitutes.
The working hypothesis that was used to evaluate this approach has been that an acellular solution can be designed to act as a universal tissue preservation solution during several hours of hypothermic whole-body washout involving cardiac arrest, with or without circulatory arrest. Under this hypothesis, Taylor et al. have formulated and evaluated two solutions designated Hypothermosol™-purge (HTS-P) and Hypothermosol™-maintenance (HTS-M) that fulfill separate requirements during the asanguineous procedure. Some aspects of these solutions are described in U.S. Pat. Nos. 5,405,742 and 5,514,536 to Taylor, both of which are incorporated herein by reference in their entireties. The Taylor patents disclose methods in which some embodiments of the compositions of this invention can be applied. Also, the Taylor patents disclose compositions that may be used in methods and kits of this invention.
The principal solution (HTS-M) is a hyperkalemic “intracellular” solution specifically designed to “maintain” cellular integrity during the hypothermic interval at the lowest temperature. The second solution is designed to interface between the blood and the HTS-M maintenance solution during both cooling and warming. This companion solution is, therefore, an “extracellular” flush solution designed to aid in purging the circulation of blood during cooling since the removal of erythrocytes from the microvasculature is an important objective during ultraprofound hypothermia. The “purge” solution is also designed to flush the system (vasculature and CPB circuit) of the hyperkalemic HTS-M solution during warming and possibly help to flush-out accumulated toxins and metabolic byproducts that might promote oxidative stress and free radical injury upon reperfusion.
Based upon the principles that have emerged from isolated organ preservation studies, an attempt was made to incorporate some of the important characteristics in the formulation of the Hypothermosol™ solutions and, wherever possible, components that might fulfill multiple roles were selected. This strategy maximizes the intrinsic qualities of the solutions that, by design as universal tissue preservation solutions, would inevitably be a hybrid of other hypothermic perfusates and storage media.
The composition of the Hypothermosol™ blood substitutes and the rationale for their formulation are discussed in U.S. Pat. Nos. 5,405,742 and 5,514,536 to Taylor, both of which are incorporated herein by reference in their entireties. These solutions have been shown to protect the brain, heart and visceral organs during 3.5 hours of cardiac arrest and global ischemia in an asanguineous canine model during controlled profound hypothermia at <10°C. Successful application of this technique to man would provide more than a 3-fold extension of the current limits of <1 hour for “safe” arrest without a high risk of neurological complications. This novel approach to bloodless surgery would significantly broaden the window of opportunity for surgical intervention in a variety of currently inoperable cases, principally in the areas of cardiovascular surgery, neurosurgery and emergency trauma surgery.
More recently, the Hypothermosol™-maintenance solution has been used for in vitro hypothermic preservation of a variety of tissues and organs including isolated hearts, fetal spinal cord and engineered skin.